Current methods and kits for preparation of specimens, especially for protection, isolation and alteration of specimens and biomolecules contains some major and key components such as the chemicals in high concentration, toxic organic reagents, and solid phase binding materials. There are many methods and kits for a great variety of specimens and biomolecules. For examples, in aspects of protection of specimens and biomolecules, using ammonium sulfate in high concentration to protect integrity of RNA (U.S. Pat. No. 6,204,375); using toxic formaldehyde to protect histological structure and micromorphology of tissue specimens. In aspects of isolation of biomolecules from specimens, using organic reagents such as phenol and chloroform to isolate RNA (U.S. Pat. No. 5,346,994); using solid phase binding materials to isolate biomolecules; using many different methods and kits to isolate biomolecules including DNA/ccfDNA, Large RNA/mRNA/ccfRNA, Small RNA/miRNA/ccfmiRNA, Protein, Lipid, Carbohydrates, and Metabolite from the same specimen; and using many different methods and kits to isolate biomolecules from a variety of specimens including solid specimens, liquid specimens, chemical or enzymatic reaction specimens, gel slice specimens containing biomolecules, and contaminated or over-diluted specimens.
But, there are some drawbacks in current methods and kits for preparation of specimens. The too many methods and kits are individually separated, limited to and focus specifically for a particular function and application. While in protection of specimens and biomolecules, ammonium sulfate in high concentration can protect integrity of RNA, but cannot protect the histological structure and micromorphology of tissue specimens; toxic formaldehyde can protects histological structure and micromorphology of tissue specimens but can also cause the degradation of biomolecule inside protected specimens. While in isolation of biomolecules from specimens, organic reagents such as phenol and chloroform are toxic to users and environment; solid phase binding materials waste economical and natural resources. While in isolation of different biomolecules from the same specimen, it demands large amount of specimen and uses many different methods and kits for different biomolecules. While in isolation of biomolecules from the different specimen, it uses many different methods and kits for a variety of specimens. The many current methods and kits for preparation of specimens are not designed for integration, interaction or sharing each other. There is almost no possibility to establish a core method and kit to cover most functions and applications in preparation of specimen and to extend the extra functions and applications to many other particular specimens and biomolecules.
Although there are many years of application and continuous researches in current methods and kits for preparation of specimens, there are still too many of current methods and kits for preparation of different specimens and biomolecules, due to the fact that each individual method and kit only use the optimized chemicals and materials particularly for the specific function and application of the method and kit itself, and no or less effort is focused on integration, interaction and sharing the components, functions and applications among the current methods and kits. Up to date, it is not available for chemicals, methods and kits that can protect the histological structure and micromorphology of tissue specimens and also protect integrity of biomolecules; it is not available for chemicals, methods and kits that can protect the protect integrity of biomolecules and also isolate biomolecules; it is not available for chemicals, methods and kits that isolate biomolecules directly from protection agents for specimens and biomolecules. It is not available for chemicals, methods and kits that can remove toxic chemicals or reagents for protection and isolation of specimens and biomolecules. Duo to incompatibility among the chemicals, methods and kits aforementioned, it is not available for a method and kit that can integrate different methods and kits that can prepare different specimens and different biomolecules; it is not available for a method and kit that can isolate biomolecules including DNA/ccfDNA, Large RNA/mRNA/ccfRNA, Small RNA/miRNA/ccfmiRNA, protein, lipid, carbohydrate, and metabolite simultaneously from the same specimen; it is not available for a method and kit that can isolate biomolecules from a variety of specimens including solid specimens, liquid specimens, chemical or enzymatic reaction specimens, gel slice specimens containing biomolecules, and contaminated or over-diluted specimens by itself; it is not available for a method and kit that can serve as a core method and kit to cover most functions and applications in preparation of specimen and to extend the extra functions and applications to many other particular specimens and biomolecules including isolation of biomolecules from formalin fixed and paraffin embedded (FFPE) tissue sections, isolation of mitochondria DNA and nucleic DNA from mitochondria and nuclei, isolation of plasmid DNA from bacteria, elimination of DNA from RNA, conversion of DNA for methylation analysis, preparation of DNA fragments, DNA labeling and RNA labeling. Therefore, current methods and kits still use toxic chemicals that hurt users and environment. More and more methods and kits with different chemicals and formats for preparation of different specimens and different biomolecules are existed and coming out continuously that bother the users' selections for choosing the methods and kits, and waste the economical and natural resources.
This invention is created for overcoming the drawbacks current methods and kits for preparation of specimens aforementioned and provides a method and kit for preparation of specimens and biomolecules. The invention provides the methods and kits for preparation of specimens and biomolecules as follows: the methods and kits for preparation of specimens and biomolecules without involving phenol and chloroform; One solution that can integratively protect, isolate and alter the specimens and biomolecules and subsequently isolate biomolecules directly from the specimens in the protection agents for specimens and biomolecules; One method and kit can versatilely isolate biomolecules from a variety of specimens including solid specimens, liquid specimens, chemical or enzymatic reaction specimens, gel slice specimens containing biomolecules, and contaminated or over-diluted specimens by itself; One method and kit can systematically isolate biomolecules including DNA/ccfDNA, Large RNA/mRNA/ccfRNA, Small RNA/miRNA/ccfmiRNA, protein, lipid, carbohydrate, and metabolite simultaneously from the same specimen; One method and kit can serve as a core method and kit to cover most functions and applications in preparation of specimen and to extend the extra functions and applications to many other particular specimens and biomolecules including isolation of biomolecules from formalin fixed and paraffin embedded (FFPE) tissue sections, isolation of mitochondria DNA and nucleic DNA from mitochondria and nuclei, isolation of plasmid DNA from bacteria, elimination of DNA from RNA, conversion of DNA for methylation analysis, preparation of DNA fragments, DNA labeling and RNA labeling.
This invention of Integrated Versatile and Systems Preparation of Specimens (Samples) relates an open module technology and system which integrates synchronously the methods of protection, isolation and alteration of specimens into one product or kit. The “One for All” product or kit comprises a core module without or with plug-in modules and a set of comprehensive protocols for systems preparation of biomolecules including DNA/ccfDNA, Large RNA/mRNA/ccfRNA, Small RNA/miRNA/ccfmiRNA, Protein, Lipid, Carbohydrates, and Metabolite simultaneously or individually from a variety of specimens including solid specimens (animal tissue, cell, plant tissue, and bacteria) and liquid specimens (plasma, serum, whole blood, biofluid).
The core module can accept and adopt new or custom Plug-in modules for expanding the functions or applications for Integrated Versatile and Systems Preparation of Specimen through open module technology. The systems biomolecules prepared from Integrated Versatile and Systems Preparation of Specimens have broad applications in systems pathology, systems diagnostics, systems medicine, systems biology and associated disciplines, as well as in conventional biomedical disciplines. The product or kit supported by the open module technology and system prepares biomolecules with high quality with the features and benefits of easy to use, fast, no toxic materials, safe to user and environment, low demanding on the materials and labors, sharing modules among users, cost-effective, saving budget, reducing waste, saving nature resources and environment and leading to a low-carbon and Green economy in preparation of specimens.
The terminology of systems preparation of biomolecules in this invention derives from Systems theory. Systems theory is a trans-disciplinary approach that abstracts and considers a system as a set of independent and interacting parts. Systems Pathology, systems diagnostics, systems medicine and systems biology are the terms used to describe a number of applications and studies in life science. They are life science based inter-disciplinary application and research fields that focus on complex interactions in life systems, claiming that they use holism perspectives instead of reductive perspectives. For examples, in reductive perspective, the accountable biomarkers for conventional diagnostics of diseases are pursued down to the details as a single nucleotide polymorphism (SNP) in genomic DNA, a change of expression pattern in one mRNA, or a reconfiguration of protein molecule; One amino acid change in a protein is used as diagnostic marker but without consideration of changes in mRNA and DNA as cause of the diseases, or changes in lipid, carbohydrate or metabolite as results of the diseases. In systems diagnostics with holism perspective, the amino acid change in a protein is considered as one part of the disease, the mRNA expression and the DNA was studied for the cause of disease, the changes in lipid, carbohydrate or metabolite are studies for the results of the disease; The cause of the disease can be in the immune system and symptoms are from neural system, digest system or urinary system.
With systems preparation of specimens, the disease was studied in inter-disciplinary field such as molecular biology, biochemistry, physiology or pathology. The complex interactions in life systems involve neural system, digest system and urinary system. With the systems diagnostics on the biomolecules of DNA, mRNA, miRNA, Protein, lipid, carbohydrate or metabolite, the approaches of systems medicine, such as therapeutics, treatment, prevention, prediction, prognostication, and participation, are used to deal with the disease. Unfortunately, the biomolecules isolated individually, irrespectively, irrelevantly, randomly, or asynchronously by conventional preparation methods are hardly fulfill the demands on systems biomolecules for systems diagnostics. Therefore, the systems preparations of systems biomolecules including DNA/ccfDNA, Large RNA/mRNA/ccfRNA, Small RNA/miRNA/ccfmiRNA, Protein, lipid, carbohydrate or metabolite simultaneously (or individually if necessary) from the specimens by this invention are very critical for systems diagnostics and systems medicine.
In this invention Integrated Preparation of Specimens integrates synchronously the three methods of preparation, protection, isolation and alteration of specimen and biomolecules in one product or Kit. Conventionally, the preparation methods of protection, isolation and alteration are carried out separately, individually, irrelevantly, irrespectively or asynchronously in different product or kit. Such a complicated mission was achieved through open module technology in this invention. The open module technology includes three major elements, the core module, the plug-in modules, and emerging and custom plug-in modules. The core module plays the leading role and major functions/applications of the Integrated Versatile and Systems Preparation of Specimens. The plug-in modules extend and broaden the functions/applications of the core module. The plug-in modules are designed and act as plug-and-play modules for the core modules. The core module is designed to accept and adopt emerging and custom plug-in modules, which allows inventor to add more plug-in modules or users to add their special plug-in modules with custom requests. The product or kit derived from the core module has multiple functions/applications of many conventional products or Kits. With products or kits derived from plug in modules the core module has a vast variety of functions/applications in almost all conventional products or Kits.
For examples, each conventional kit for specimen preparation only specifically prepares one type of biomolecule, such as DNA isolation Kit for preparation of DNA only. Different types of conventional kits for specimen preparation are required for preparation different types of biomolecules, such as RNA isolation kit, miRNA isolation kit and protein isolation kit. The protection of specimen is carried out by other kit, such as tissue preservation kit, and alteration of biomolecules will be conducted by another kit, such as DNA conversion kit for methylation analysis. Total six kits are required for above jobs of protection, isolation and alteration. In this invention one kit with core module and one plug-in module will complete these jobs. If users want to prepare circulating cell free (ccf) DNA, ccfRNA and ccfmiRNA from plasma for their project, another three kits are required if they use conventional kits, which accumulates to total nine kits for nine jobs. But with this invention, they can use the extra functions of the same kit for prepare ccfDNA, ccfRNA and ccfmiRNA from plasma. The one kit in this invention is same as nine conventional kits in this example. The Integrated Versatile and Systems Preparation of Specimens integrates the complicated missions with many different kits into state of art, One Kit for All.